甘蓝型油菜隐性杂合两型系SLAB育性候选基因的筛选鉴定

发布时间：2018-06-08 10:58

本文选题：甘蓝型油菜 + 隐性核不育　；参考：《西南大学》2017年硕士论文

【摘要】：甘蓝型油菜是世界上重要的油料作物,利用隐性雄性不育授粉系统制备强优势杂交种是甘蓝型油菜育种的重要内容。本研究通过石蜡切片和扫描电镜对甘蓝型油菜隐性杂合两型系SLA和SLB花药发育的特点进行细胞学分析,初步确定不育系SLA发生败育的类型和时期。利用NGS技术对两型系SLA和SLB进行了转录组和基因组重测序分析,在基因组水平上分析SLA和SLB之间的遗传变异;用转录组和qRT-PCR方法筛选在SLA和SLB中的差异表达基因;应用在线分析软件Venny比较分析转录组差异表达基因和重测序发生变异的基因,确定与花药败育相关的候选基因。其主要结果如下:扫描电镜分析结果显示:不育系SLA的花药显著小于可育系SLB,整个发育过程花药无明显的伸长生长。当花蕾大小为2mm时,不育系SLA和可育系SLB花药表层细胞无显著差异;花蕾大小为4mm时,不育系SLA花药表层细胞开始塌陷萎缩,背面细胞排列紧密;当花蕾开放后,不育系SLA花药细胞完全萎缩、退化,花粉囊不能正常开裂释放花粉。不同发育期花蕾石蜡切片甲苯胺蓝染色的细胞学结果显示:在四分体时期,不育系SLA和可育系SLB的花药存在显著差异,SLA的绒毡层细胞高度液泡化,不能皱缩、转化为分泌型;随后在小孢子发育过程中逐渐降解消失,不能正常发育为成熟小孢子。因此,不育系SLA发生败育主要原因可能是在四分体时期或四分体之前绒毡层细胞的发育异常而导致。通过对两型系SLA和SLB花蕾转录组数据比较分析,共获得101个在不育材料SLA中显著上调表达的差异基因。采用GO功能分类将三个类别中已注释基因划分为25个功能类别。101个差异表达基因中,在分子功能方面富集最多,有55个基因发生富集,主要在离子结合、电子载体活性等能量代谢相关代谢过程;在生物过程方面有44个基因发生富集,主要在碳水化合物代谢、蛋白水解、抗氧化反应等条目中;在细胞成分方面富集到2个基因,主要在胞外部分的条目中,说明甘蓝型油菜育性调控是一个复杂的生物过程。KEGG代谢途径分析发现,差异表达基因仅在苯丙烷代谢途径中显著富集,共有4个显著差异表达基因,包括1个ACOS5基因(BnaC09g10950D)和3个过氧化物酶家族基因(BnaA01g01280D、BnaA07g01880D、BnaC01g02300D),说明苯丙烷代谢途径在不育系SLA发生败育过程中起重要作用。通过对SLA和SLB全基因组重测序数据比较分析,在整个基因组中检测到119,863个SNP位点和18,370个Indel变异位点,这些变异位点大部分发生在基因间或内含子等非编码区域,在各条染色体上的分布存在差异性。根据变异位点在参考基因组上对应的基因物理位置,共注释到影响基因表达或者功能的错义突变、移码突变等变异的基因14,705个;同时针对这些变异基因进行GO功能富集分析,发现这些基因主要在核苷结合、碳水化合物衍生物结合、有机物代谢过程通路等条目发生富集。综合转录组和全基因组重测序结果,共检测到11个育性关键差异表达基因,其中3个基因(BnaA03Rf-4、BnaA10Rf-7、BnaC05Rf-10)存在2种以上位点变异类型。另外,在内含子区域发生变异的基因有4个(BnaA03Rf-3、BnaA04Rf-5、BnaA10Rf-6、BnaAnnRf-8),发生同义突变基因2个(BnaA03Rf-4、BnaC03Rf-9),在上游或下游区域发生变异的有6个基因(BnaA01Rf-1、BnaA02Rf-2、BnaA03Rf-4、BnaA10Rf-7、BnaC05Rf-10、BnaC07Rf-11)。qRT-PCR结果显示11个基因中有8个基因在SLA的Bu1(1 mm花蕾)中均显著上调表达,qRT-PCR验证的这8个基因可作为关键育性候选基因。
[Abstract]:Brassica napus is an important oil crop in the world. Using the Recessive Male Sterile pollination system to prepare strong heterosis is an important content of Brassica napus breeding. In this study, the characteristics of the anther development of SLA and SLB in the recessive heterozygous line of Brassica napus were analyzed by paraffin section and scanning electron microscope. The type and period of abortion in line SLA, the transcriptional and genomic resequencing analysis of SLA and SLB in type two lines was carried out by NGS technology, the genetic variation between SLA and SLB was analyzed at the genomic level, and the differential expression genes in SLA and SLB were screened by the transcriptional group and qRT-PCR method, and the transcriptional analysis was compared with the online analysis software Venny. The results of the scanning electron microscope analysis showed that the anther of SLA was significantly smaller than that of the fertile line SLB, and the anther was not elongated in the whole development process. When the bud size was 2mm, the sterile line SLA and the fertile line SLB were found. When the size of buds was 4mm, the surface cells of the sterile line SLA anther began to collapse and atrophy and the back cells were closely arranged. When the buds were open, the SLA anther cells of the sterile line were completely atrophied and degenerated. The pollen sac could not normally crack and release pollen. The cytological junction of the paraffin section of the bud in different development stages was stained with toluidine blue. The results showed that there were significant differences in anthers between the sterile line SLA and the fertile line SLB during the four division. The tapetum cells in SLA were highly vacuolated, and they could not be crinkled and converted to secretory type. Then, the degradation of the microspore was gradually disappearing and could not normally develop into mature microspore. Therefore, the main cause of abortion of the sterile line SLA may be in the process of abortion. The development of tapetum cells was abnormal before or before the four division or four division. Through comparison and analysis of the data of the SLA and SLB buds transcriptional group of the two type system, 101 differentially expressed genes were significantly up-regulated in the sterile material SLA. The GO functional classification was used to divide the annotated genes in the three categories into 25 functional categories.101 difference tables In the gene, the most abundant gene is enriched in the molecular function, and 55 genes are enriched, mainly in the energy metabolism related metabolic processes, such as ion binding and the activity of electron carrier. In the biological process, 44 genes are enriched, mainly in carbohydrate metabolism, protein hydrolysis, antioxidant reaction and so on. The enrichment of 2 in the cell composition is 2. A gene, mainly in the entry of the extracellular part, shows that the fertility regulation of Brassica napus is a complex biological process.KEGG metabolic pathway analysis. The differentially expressed genes are enriched only in the phenylpropane metabolic pathway, and there are 4 significant differentially expressed genes, including 1 ACOS5 genes (BnaC09g10950D) and 3 peroxidase families. Gene (BnaA01g01280D, BnaA07g01880D, BnaC01g02300D), indicating that the phenylpropane metabolic pathway plays an important role in the abortion process of the sterile line SLA. Through the comparative analysis of the whole genome resequencing data of SLA and SLB, 119863 SNP loci and 18370 Indel mutation sites are detected in the whole genome. Most of these mutation sites occur. In non coding regions such as the INTERGENE or intron, there are differences in the distribution of the chromosomes. According to the location of the genes corresponding to the reference genome, 14705 mutations, such as the missense mutation and the shift mutation, which affect the gene expression or function, are also annotated, and the GO function for these variants is performed at the same time. Enrichment analysis showed that these genes were mainly enriched in nucleoside binding, carbohydrate derivative binding, and organic matter metabolic pathway pathway, and 11 key differentially expressed genes were detected in the comprehensive transcriptional group and total genome resequencing, of which 3 genes (BnaA03Rf-4, BnaA10Rf-7, BnaC05Rf-10) had 2 kinds of epistasis In addition, 4 genes (BnaA03Rf-3, BnaA04Rf-5, BnaA10Rf-6, BnaAnnRf-8) were mutated in the intron region, and 2 (BnaA03Rf-4, BnaC03Rf-9) of the synonymous mutation (BnaA03Rf-4, BnaC03Rf-9) were occurring, and 6 genes (BnaA01Rf-1, BnaA02Rf-2, BnaA03Rf-4, BnaA10Rf-7, BnaC05Rf-10, BnaC07Rf-11) occurred in the upstream or downstream regions. The results showed that 8 of the 11 genes were significantly up-regulated in Bu1 (1 mm buds) of SLA, and these 8 genes verified by qRT-PCR could be used as key fertility candidate genes.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4

【参考文献】